Despite the introduction of highly active antiretroviral therapy (HAART), HIV associated neurocognitive disorder (HAND) remains a significant cause of morbidity. Previous work has demonstrated that HAND is related to the severity of HIV associated neurodegeneration. In preliminary microarray work we have noted that the gene expression of a number of proteins related to the innate immune system toll-like receptor (TLR) signaling pathway correlates strongly with HIV associated neurodegeneration, most significantly TLR3 and -4. We have further demonstrated that treatment of astrocytes with gp120 (Bal) resulted in an increase in expression of TLR4. In the current application we wish to examine the potential relationship between TLR3 and 4 gene expression and exposure to HIV. TLR3 and -4 expressions will be assessed initially in primary human neuroglial cultures exposed to supernatants taken from HIV infected (Bal and SF162) monocyte derived macrophages (MDMs). mRNA expression will be measured via quantitative real time polymerase chain reaction (qRT-PCR) with protein expression measured via immunoblot and immunohistochemistry (IHC). In the second part of this proposal we will investigate the effects of blocking TLR3 and -4 expressions on HIV associated neurotoxicity. Similar to specific aim 1 and using the data generated from this aim we will treat primary human neuronal cultures with supernatants from HIV infected (Bal and SF162) MDMs at concentrations and time-points shown to cause toxicity. This will be in the absence and presence of siRNA's to TLR3 and -4. We will then verify this knockdown using qRT-PCR and assess the effects of this on HIV neurotoxic effects. As a potential mechanism for TLR induced neurodegeneration we will assess GSK3b activity in a final set of experiments using ELISA and also the ability of specific GSK3b inhibitors A014418 and B6B30 to prevent or attenuate HIV associated neurotoxicity. Data generated from this proposal will provide a first step towards elucidating the mechanism by which TLR gene products result in HIV associated neurodegeneration and also identify potential novel targets for ameliorating this toxic process. PUBLIC HEALTH RELEVANCE The ability to reduce or prevent neuronal loss associated with HIV has the potential to prevent or attenuate the effects of HAND. The focus of this proposal is to further characterize the role of TLR 3 and 4 dysregulation in HIV associated neurodegeneration and contribution of GSK3b activity in regulating this mechanism. Inhibition of TLR 3 and 4 activity may present a possible way to prevent HIV associated neurodegeneration and therefore alleviate symptoms of HAND.